Mortality from colorectal cancer (CRC) is significant, and novel CRC therapies are needed. A pseudokinase MLKL typically executes necroptotic cell death, and MLKL inactivation protects cells from such death. However, we found unexpectedly that MLKL gene knockout enhanced CRC cell death caused by a protein synthesis inhibitor homoharringtonine used for chronic myeloid leukemia treatment. In an effort to explain this finding, we observed that MLKL gene knockout reduced CRC cell autophagy and rendered such autophagy critically dependent on the presence of VPS37A, a component of the ESCRT-I complex. Moreover, homoharringtonine-induced activation of p38 MAP kinase (p38MAPK) prevented VPS37A from supporting autophagy in MLKL-deficient cells and triggered their parthanatos, a cell death type driven by poly(ADP-ribose) polymerase hyperactivation. Finally, a pharmacological MLKL inhibitor necrosulfonamide strongly cooperated with homoharringtonine in suppressing CRC cell tumorigenicity in mice. Thus, while MLKL mediates necroptosis, MLKL protects CRC cells from death caused by drugs blocking basal autophagy, e.g., homoharringtonine, and MLKL inhibition creates a therapeutic vulnerability that could be utilized for CRC treatment.
{"title":"Inactivation of necroptosis-promoting protein MLKL creates a therapeutic vulnerability in colorectal cancer cells.","authors":"Peijia Jiang, Sandhya Chipurupalli, Byong Hoon Yoo, Xiaoyang Liu, Kirill V Rosen","doi":"10.1101/2024.09.05.611491","DOIUrl":"https://doi.org/10.1101/2024.09.05.611491","url":null,"abstract":"Mortality from colorectal cancer (CRC) is significant, and novel CRC therapies are needed. A pseudokinase MLKL typically executes necroptotic cell death, and MLKL inactivation protects cells from such death. However, we found unexpectedly that MLKL gene knockout enhanced CRC cell death caused by a protein synthesis inhibitor homoharringtonine used for chronic myeloid leukemia treatment. In an effort to explain this finding, we observed that MLKL gene knockout reduced CRC cell autophagy and rendered such autophagy critically dependent on the presence of VPS37A, a component of the ESCRT-I complex. Moreover, homoharringtonine-induced activation of p38 MAP kinase (p38MAPK) prevented VPS37A from supporting autophagy in MLKL-deficient cells and triggered their parthanatos, a cell death type driven by poly(ADP-ribose) polymerase hyperactivation. Finally, a pharmacological MLKL inhibitor necrosulfonamide strongly cooperated with homoharringtonine in suppressing CRC cell tumorigenicity in mice. Thus, while MLKL mediates necroptosis, MLKL protects CRC cells from death caused by drugs blocking basal autophagy, e.g., homoharringtonine, and MLKL inhibition creates a therapeutic vulnerability that could be utilized for CRC treatment.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142210529","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-06DOI: 10.1101/2024.09.05.611456
Mairead Commane, Vidula Jadhav, Katerina Leonova, Henry Withers, Brian Buckley, Katerina Gurova
The phenotypic plasticity of cancer cells has recently emerged as an important factor of treatment failure. The mechanisms of phenotypic plasticity are not fully understood. One of the hypotheses is that the degree of chromatin accessibility defines the easiness of cell transitions between different phenotypes. To test this, a method to compare overall chromatin accessibility between cells in a population or between cell populations is needed. We propose to measure chromatin accessibility by fluorescence signal from nuclei of cells stained with DNA binding fluorescent molecules. This method is based on the observations that small molecules bind nucleosome-free DNA more easily than nucleosomal DNA. Thus, nuclear fluorescence is proportional to the amount of nucleosome-free DNA, serving as a measure of chromatin accessibility. We optimized the method using several DNA intercalators and minor groove binders and known chromatin-modulating agents and demonstrated that chromatin accessibility is increased upon oncogene-induced transformation and further in tumor cells.
最近，癌细胞的表型可塑性已成为治疗失败的一个重要因素。表型可塑性的机制尚不完全清楚。其中一个假设是，染色质可及性的程度决定了细胞在不同表型之间转换的难易程度。为了验证这一点，需要一种方法来比较群体中细胞之间或细胞群体之间的整体染色质可及性。我们建议通过 DNA 结合荧光分子染色细胞核的荧光信号来测量染色质的可及性。这种方法基于小分子比核糖体 DNA 更容易结合无核糖体 DNA 的观察结果。因此，细胞核荧光与无核糖体 DNA 的数量成正比，可作为染色质可及性的测量指标。我们利用几种DNA插入因子和小沟结合剂以及已知的染色质调节剂对该方法进行了优化，并证明染色质可及性在癌基因诱导的转化过程中会增加，并在肿瘤细胞中进一步增加。
{"title":"Image-Based Quantitative Single-Cell Method Showed Increase of Global Chromatin Accessibility in Tumor Compared to Normal Cells","authors":"Mairead Commane, Vidula Jadhav, Katerina Leonova, Henry Withers, Brian Buckley, Katerina Gurova","doi":"10.1101/2024.09.05.611456","DOIUrl":"https://doi.org/10.1101/2024.09.05.611456","url":null,"abstract":"The phenotypic plasticity of cancer cells has recently emerged as an important factor of treatment failure. The mechanisms of phenotypic plasticity are not fully understood. One of the hypotheses is that the degree of chromatin accessibility defines the easiness of cell transitions between different phenotypes. To test this, a method to compare overall chromatin accessibility between cells in a population or between cell populations is needed. We propose to measure chromatin accessibility by fluorescence signal from nuclei of cells stained with DNA binding fluorescent molecules. This method is based on the observations that small molecules bind nucleosome-free DNA more easily than nucleosomal DNA. Thus, nuclear fluorescence is proportional to the amount of nucleosome-free DNA, serving as a measure of chromatin accessibility. We optimized the method using several DNA intercalators and minor groove binders and known chromatin-modulating agents and demonstrated that chromatin accessibility is increased upon oncogene-induced transformation and further in tumor cells.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142210501","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-06DOI: 10.1101/2024.09.06.611619
Ludivine Bertonnier-Brouty, Kavya Achanta, Jonas Andersson, Sara Bsharat, Tania Singh, Tuomas Kaprio, Jaana Hagstrom, Caj Haglund, Hanna Seppanen, Rashmi B Prasad, Isabella Artner
Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer lacking effective drugs and therefore new treatment targets are needed. Transcriptomic analysis comparing human embryonic and PDAC tissue identified a large overlap of expression profiles suggesting a re-initiation of developmental programs in pancreatic cancer. Specifically, we identified the transcription factors HOXB6 and HOXB8 as potential key regulators in PDAC. Loss of HOXB6 and HOXB8 in pancreatic cancer cells inhibited cell proliferation, induced apoptosis and senescence and enhanced gemcitabine sensitivity. Moreover, reduced HOXB6 and HOXB8 expression in pancreatic and lung adenocarcinoma cell lines affected transcription of immune response pathways which resulted in an increased sensitivity of cancer cells to anti-tumorigenic activities of macrophages suggesting that the HOXB6 and HOXB8 immune regulatory pattern is conserved in different cancer types. Additionally, naive M0 macrophages exposed to HOXB8 deficient PDAC cells were unable to differentiate into tumor associated macrophages, suggesting that HOXB8 promotes the transition of initial anti-tumor macrophage to a tumor-promoting macrophage phenotype in pancreatic cancer. Our findings indicate that HOXB6 and HOXB8 play important roles in regulating cell proliferation, immune response and treatment resistance to promote pancreatic cancer tumorigenesis and could be useful therapeutic targets.
{"title":"HOXB6 and HOXB8 control immune-cancer cell interactions in pancreatic cancer.","authors":"Ludivine Bertonnier-Brouty, Kavya Achanta, Jonas Andersson, Sara Bsharat, Tania Singh, Tuomas Kaprio, Jaana Hagstrom, Caj Haglund, Hanna Seppanen, Rashmi B Prasad, Isabella Artner","doi":"10.1101/2024.09.06.611619","DOIUrl":"https://doi.org/10.1101/2024.09.06.611619","url":null,"abstract":"Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer lacking effective drugs and therefore new treatment targets are needed. Transcriptomic analysis comparing human embryonic and PDAC tissue identified a large overlap of expression profiles suggesting a re-initiation of developmental programs in pancreatic cancer. Specifically, we identified the transcription factors HOXB6 and HOXB8 as potential key regulators in PDAC. Loss of HOXB6 and HOXB8 in pancreatic cancer cells inhibited cell proliferation, induced apoptosis and senescence and enhanced gemcitabine sensitivity. Moreover, reduced HOXB6 and HOXB8 expression in pancreatic and lung adenocarcinoma cell lines affected transcription of immune response pathways which resulted in an increased sensitivity of cancer cells to anti-tumorigenic activities of macrophages suggesting that the HOXB6 and HOXB8 immune regulatory pattern is conserved in different cancer types. Additionally, naive M0 macrophages exposed to HOXB8 deficient PDAC cells were unable to differentiate into tumor associated macrophages, suggesting that HOXB8 promotes the transition of initial anti-tumor macrophage to a tumor-promoting macrophage phenotype in pancreatic cancer. Our findings indicate that HOXB6 and HOXB8 play important roles in regulating cell proliferation, immune response and treatment resistance to promote pancreatic cancer tumorigenesis and could be useful therapeutic targets.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":"85 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142210522","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-06DOI: 10.1101/2024.09.04.611258
Mateusz Garbulowski, Riccardo Mosca, Carlos J. Gallardo-Dodd, Claudia Kutter, Erik L. L. Sonnhammer
RNA binding proteins (RBPs) are essential components of the transcriptomic regulome. Identifying the RBP regulome in cancer cells is crucial to discovering and understanding carcinogenesis mechanisms and providing new therapeutic targets. Here, we aimed to reveal the regulome of liver cancer upon specific perturbations. To this end, we applied a consensus Gene Regulatory Network (GRN) approach using knockdown data for the liver cancer cell line HepG2. By incorporating multiple GRNs from diverse inference methods, we constructed a highly precise GRN. To validate our results, we comprehensively evaluated the consensus GRN, focusing on characterizing the most relevant aspects of the liver cancer regulome. This included utilizing eCLIP-seq and RAPseq data to verify RBP interactions and binding sites. In addition, we performed an enrichment analysis of network modules and drug repurposing based on the inferred GRN. Taken together, our findings demonstrate the critical roles of RBP regulatory interactions in liver cancer that can be employed to improve treatment strategies.
{"title":"Comprehensive analysis of the RBP regulome reveals functional modules and drug candidates in liver cancer","authors":"Mateusz Garbulowski, Riccardo Mosca, Carlos J. Gallardo-Dodd, Claudia Kutter, Erik L. L. Sonnhammer","doi":"10.1101/2024.09.04.611258","DOIUrl":"https://doi.org/10.1101/2024.09.04.611258","url":null,"abstract":"RNA binding proteins (RBPs) are essential components of the transcriptomic regulome. Identifying the RBP regulome in cancer cells is crucial to discovering and understanding carcinogenesis mechanisms and providing new therapeutic targets. Here, we aimed to reveal the regulome of liver cancer upon specific perturbations. To this end, we applied a consensus Gene Regulatory Network (GRN) approach using knockdown data for the liver cancer cell line HepG2. By incorporating multiple GRNs from diverse inference methods, we constructed a highly precise GRN. To validate our results, we comprehensively evaluated the consensus GRN, focusing on characterizing the most relevant aspects of the liver cancer regulome. This included utilizing eCLIP-seq and RAPseq data to verify RBP interactions and binding sites. In addition, we performed an enrichment analysis of network modules and drug repurposing based on the inferred GRN. Taken together, our findings demonstrate the critical roles of RBP regulatory interactions in liver cancer that can be employed to improve treatment strategies.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142210526","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-06DOI: 10.1101/2024.09.05.611436
Patricia Eror Barnes, Maria Jose de la Concha, Kioko Mwikali, Bee Ling Ng, Hannes Ponstingl, Alena Pance
The fundamental transcription factor p53 regulates cellular processes and integrates signals of cellular stress, triggering a coordinated response to ensure survival of cells restored to healthy function and programmed death of those that could not be repaired. Unsurprisingly, this is one of the most mutated genes in human cancers, with most changes occurring in the DNA-binding domain of the protein. In this work, we take a genome-wide approach and use available resources to identify high confidence p53-target genes, that we examine in three breast cancer cell lines with different p53 status, wild type (MCF-7) and different mutations in the DNA-binding domain (MDA-MB231, T47D). Comparison of p53-targets expression in response to DNA damage by RNAseq and cellular assays reveals that MDA-MB231 have a severely impaired p53-dependent pathway functionality while T47D are much less affected. MDA-MB231 are more resistant to DNA damage yet unable to repair and able to override cell cycle arrest leading to survival while T47D are sensitive only to high dose and exposure to genotoxic agents. This data shows the variability of effects of different p53 mutations and highlight the importance of understanding the mechanisms of p53 in the context of genotoxicity-based treatment.
基本转录因子 p53 可调节细胞过程并整合细胞压力信号，引发协调反应，确保恢复健康功能的细胞存活，并使无法修复的细胞按程序死亡。毫不奇怪，这是人类癌症中突变最多的基因之一，大多数变化发生在蛋白质的 DNA 结合域。在这项工作中，我们采用全基因组方法，利用现有资源确定了高置信度的 p53 靶基因，并在三个具有不同 p53 状态的乳腺癌细胞系（野生型（MCF-7）和 DNA 结合域发生不同突变的细胞系（MDA-MB231、T47D）中进行了研究。通过 RNAseq 和细胞检测比较 p53 靶标在 DNA 损伤反应中的表达，发现 MDA-MB231 的 p53 依赖性通路功能严重受损，而 T47D 受到的影响要小得多。MDA-MB231 对 DNA 损伤有更强的抵抗力，但无法进行修复，并能超越细胞周期停滞而存活，而 T47D 只对高剂量和暴露于基因毒性制剂敏感。这些数据显示了不同的 p53 突变所产生的不同影响，并强调了在基于基因毒性的治疗中了解 p53 机制的重要性。
{"title":"Differential gene expression in cells with different p53 mutations identifies genome-wide p53 targets and shows distinct modulation of cellular pathways in response to DNA damage","authors":"Patricia Eror Barnes, Maria Jose de la Concha, Kioko Mwikali, Bee Ling Ng, Hannes Ponstingl, Alena Pance","doi":"10.1101/2024.09.05.611436","DOIUrl":"https://doi.org/10.1101/2024.09.05.611436","url":null,"abstract":"The fundamental transcription factor p53 regulates cellular processes and integrates signals of cellular stress, triggering a coordinated response to ensure survival of cells restored to healthy function and programmed death of those that could not be repaired. Unsurprisingly, this is one of the most mutated genes in human cancers, with most changes occurring in the DNA-binding domain of the protein. In this work, we take a genome-wide approach and use available resources to identify high confidence p53-target genes, that we examine in three breast cancer cell lines with different p53 status, wild type (MCF-7) and different mutations in the DNA-binding domain (MDA-MB231, T47D). Comparison of p53-targets expression in response to DNA damage by RNAseq and cellular assays reveals that MDA-MB231 have a severely impaired p53-dependent pathway functionality while T47D are much less affected. MDA-MB231 are more resistant to DNA damage yet unable to repair and able to override cell cycle arrest leading to survival while T47D are sensitive only to high dose and exposure to genotoxic agents. This data shows the variability of effects of different p53 mutations and highlight the importance of understanding the mechanisms of p53 in the context of genotoxicity-based treatment.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":"177 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142210527","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-11DOI: 10.1101/2024.08.11.607471
Haoran Mu, Yining Tao, Jinzeng Wang, Xin He, Qi Zhang, Weixi Chen, Bing Yao, Sen Ding, Xiyu Yang, Liyuan Zhang, Hongsheng Wang, Dongqing Zuo, Jiakang Shen, Mengxiong Sun, Haoyu Wang, Ming Jiao, Xinmeng Jin, Yinhui Jin, Youzhi Liang, Yuyan Gong, Winfred Mao, Qian Liu, Zhuoying Wang, Yu Lv, Jing Xu, Tao Zhang, Yuqin Yang, Jun Lin, Fred J. Asward, James D. Joseph, Mingxi Li, Zhengdong Cai, Wei Sun, Liu Yang, Yingqi Hua
Osteosarcoma (OS) exhibit intra- and inter- heterogeneity, complicating the exploration of effective therapeutic strategies. Traditional in vitro and in vivo models are limited in inheriting biological and genomic heterogeneities of OS patients, even in inheriting the features on tumor microenvironment. The prolonged generation time of current models makes the drug development of OS slow and is not suitable to clinically rapid timing. Here, we introduce methods for generating and biobanking patient/PDX-derived osteosarcoma organoids (OS PD(X)Os) that recapitulate the histological, biological and genomic features of their paired OS patients. OS PD(X)Os can be generated quickly with high reliability in vitro or transplanted to immunodeficient mice. We further demonstrate an immune-featured OS PD(X)O (named iOS) model and its method for testing personalized chemotherapy response, personalized immune therapeutic strategy and target drug development, such as a novel PRMT5MTA inhibitor ARPN2169 on MTAP-deleted OS. Our studies show that iOS models maintain many typical features of OS and could be rapidly employed to investigate patient-specific therapeutic strategies. Additionally, our biobank establishes a rich resource for basic, translational and even clinical OS researches.
骨肉瘤（Osteosarcoma，OS）具有瘤内和瘤间异质性，使有效治疗策略的探索变得更加复杂。传统的体外和体内模型在继承骨肉瘤患者的生物学和基因组异质性方面受到限制，甚至在继承肿瘤微环境特征方面也是如此。目前的模型生成时间较长，导致 OS 药物开发进展缓慢，不适合临床快速开发。在此，我们介绍了患者/PDX衍生骨肉瘤器官组织（OS PD(X)Os）的生成和生物库方法，这些器官组织能再现配对OS患者的组织学、生物学和基因组特征。OS PD(X)Os可在体外快速生成，可靠性高，也可移植到免疫缺陷小鼠体内。我们进一步展示了一种具有免疫特征的OS PD(X)O（命名为iOS）模型及其用于测试个性化化疗反应、个性化免疫治疗策略和靶向药物开发的方法，例如在MTAP缺失的OS上测试新型PRMT5MTA抑制剂ARPN2169。我们的研究表明，iOS 模型保持了 OS 的许多典型特征，可迅速用于研究针对患者的治疗策略。此外，我们的生物库为 OS 的基础研究、转化研究甚至临床研究提供了丰富的资源。
{"title":"A Self-Assembling Immune-Featured Osteosarcoma Patient/PDX Derived Organoid Model and Biobank for Personalized Immune Therapy","authors":"Haoran Mu, Yining Tao, Jinzeng Wang, Xin He, Qi Zhang, Weixi Chen, Bing Yao, Sen Ding, Xiyu Yang, Liyuan Zhang, Hongsheng Wang, Dongqing Zuo, Jiakang Shen, Mengxiong Sun, Haoyu Wang, Ming Jiao, Xinmeng Jin, Yinhui Jin, Youzhi Liang, Yuyan Gong, Winfred Mao, Qian Liu, Zhuoying Wang, Yu Lv, Jing Xu, Tao Zhang, Yuqin Yang, Jun Lin, Fred J. Asward, James D. Joseph, Mingxi Li, Zhengdong Cai, Wei Sun, Liu Yang, Yingqi Hua","doi":"10.1101/2024.08.11.607471","DOIUrl":"https://doi.org/10.1101/2024.08.11.607471","url":null,"abstract":"Osteosarcoma (OS) exhibit intra- and inter- heterogeneity, complicating the exploration of effective therapeutic strategies. Traditional in vitro and in vivo models are limited in inheriting biological and genomic heterogeneities of OS patients, even in inheriting the features on tumor microenvironment. The prolonged generation time of current models makes the drug development of OS slow and is not suitable to clinically rapid timing. Here, we introduce methods for generating and biobanking patient/PDX-derived osteosarcoma organoids (OS PD(X)Os) that recapitulate the histological, biological and genomic features of their paired OS patients. OS PD(X)Os can be generated quickly with high reliability in vitro or transplanted to immunodeficient mice. We further demonstrate an immune-featured OS PD(X)O (named iOS) model and its method for testing personalized chemotherapy response, personalized immune therapeutic strategy and target drug development, such as a novel PRMT5MTA inhibitor ARPN2169 on MTAP-deleted OS. Our studies show that iOS models maintain many typical features of OS and could be rapidly employed to investigate patient-specific therapeutic strategies. Additionally, our biobank establishes a rich resource for basic, translational and even clinical OS researches.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141931022","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.607029
Anutr Sivakoses, Haley Quinn Marcarian, Anika M Arias, Alice R Lam, Olivia C Ihedioha, Juan A Santamaria, Geoffrey C Gurtner, Alfred L. M. Bothwell
Trogocytosis is the process by which a recipient cell siphons small membrane fragments and proteins from a donor cell and may be utilized by cancer cells to avoid immune detection. We observed lymphocyte specific protein expressed by TNBC cells via immunofluorescence imaging of patient samples. Image analysis of CD45RA expression, a T cell specific protein, revealed that all stages of TNBCs express CD45RA. Flow cytometry revealed TNBC cells trogocytose CD45 protein from T cells. We also showed that the acquisition of these lymphoid markers is contact dependent. Confocal and super-resolution imaging further revealed CD45+ spherical structures containing T cell genomic DNA inside TNBC cells after co-culture. Trogocytosis between T cells and TNBC cells altered cancer cell gene expression. Our results revealed that CD45 is obtained by TNBC cells from T cells via trogocytosis and that TNBC cells express CD45 intracellularly and on the membrane.
{"title":"Triple Negative Breast Cancer Cells Acquire Lymphocyte Proteins and Genomic DNA During Trogocytosis with T Cells","authors":"Anutr Sivakoses, Haley Quinn Marcarian, Anika M Arias, Alice R Lam, Olivia C Ihedioha, Juan A Santamaria, Geoffrey C Gurtner, Alfred L. M. Bothwell","doi":"10.1101/2024.08.09.607029","DOIUrl":"https://doi.org/10.1101/2024.08.09.607029","url":null,"abstract":"Trogocytosis is the process by which a recipient cell siphons small membrane fragments and proteins from a donor cell and may be utilized by cancer cells to avoid immune detection. We observed lymphocyte specific protein expressed by TNBC cells via immunofluorescence imaging of patient samples. Image analysis of CD45RA expression, a T cell specific protein, revealed that all stages of TNBCs express CD45RA. Flow cytometry revealed TNBC cells trogocytose CD45 protein from T cells. We also showed that the acquisition of these lymphoid markers is contact dependent. Confocal and super-resolution imaging further revealed CD45+ spherical structures containing T cell genomic DNA inside TNBC cells after co-culture. Trogocytosis between T cells and TNBC cells altered cancer cell gene expression. Our results revealed that CD45 is obtained by TNBC cells from T cells via trogocytosis and that TNBC cells express CD45 intracellularly and on the membrane.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141931095","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.607341
Aswini Panigrahi, Allison L Hunt, Diego Assis, Matthew Willetts, Bhaskar V Kallakury, Bruce Davidson, Thomas P Conrads, Radoslav Goldman
We employed laser microdissection to selectively harvest tumor cells and stroma from the microenvironment of formalin-fixed, paraffin-embedded head and neck squamous cell carcinoma (HNSCC) tissues. The captured HNSCC tissue fractions were analyzed by quantitative mass spectrometry-based proteomics using a data independent analysis approach. In paired samples, we achieved excellent proteome coverage having quantified 6,668 proteins with a median quantitative coefficient of variation under 10%. We observed significant differences in relevant functional pathways between the spatially resolved tumor and stroma regions. Our results identified extracellular matrix (ECM) as a major component enriched in the stroma, including many cancer associated fibroblast signature proteins in this compartment. We demonstrate the potential for comparative deep proteome analysis from very low starting input in a scalable format that is useful to decipher the alterations in tumor and the stromal microenvironment. Correlating such results with clinical features or disease progression will likely enable identification of novel targets for disease classification and interventions.
{"title":"dia-PASEF Proteomics of Tumor and Stroma LMD Enriched from Archived HNSCC Samples","authors":"Aswini Panigrahi, Allison L Hunt, Diego Assis, Matthew Willetts, Bhaskar V Kallakury, Bruce Davidson, Thomas P Conrads, Radoslav Goldman","doi":"10.1101/2024.08.09.607341","DOIUrl":"https://doi.org/10.1101/2024.08.09.607341","url":null,"abstract":"We employed laser microdissection to selectively harvest tumor cells and stroma from the microenvironment of formalin-fixed, paraffin-embedded head and neck squamous cell carcinoma (HNSCC) tissues. The captured HNSCC tissue fractions were analyzed by quantitative mass spectrometry-based proteomics using a data independent analysis approach. In paired samples, we achieved excellent proteome coverage having quantified 6,668 proteins with a median quantitative coefficient of variation under 10%. We observed significant differences in relevant functional pathways between the spatially resolved tumor and stroma regions. Our results identified extracellular matrix (ECM) as a major component enriched in the stroma, including many cancer associated fibroblast signature proteins in this compartment. We demonstrate the potential for comparative deep proteome analysis from very low starting input in a scalable format that is useful to decipher the alterations in tumor and the stromal microenvironment. Correlating such results with clinical features or disease progression will likely enable identification of novel targets for disease classification and interventions.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":"88 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141931014","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.607157
Elías Vera-Sigüenza, Cristina Escribano-Gonzalez, Irene Serrano-Gonzalo, Kattri-Liis Eskla, Charlotte Speakman, Alejandro Huerta-Uribe, Lisa Vettore, Himani Rana, Adam Boufersaoui, Hans Vellama, Ramin Nashebi, Ielyaas Cloete, Jennie Roberts, Supratik Basu, Mark Drayson, Christopher Bunce, Guy Pratt, Fabian Spill, Oliver D.K. Maddocks, Daniel A. Tennant
Multiple myeloma is an incurable cancer of plasma cells that depends on the bone marrow for its survival. Despite its prevalence, the molecular mechanisms underlying this malignancy remain poorly understood. In this study, we aim to bridge this knowledge gap by elucidating the metabolic interplay between myeloma cells and bone marrow mesenchymal stem cells (BMMSCs). BMMSCs are crucial in supporting myeloma cell metabolism, contributing to their proliferation, survival, and resistance to chemotherapy. Through a combination of mathematical modelling and experimental co-cultures, we demonstrate that pyruvate – the end product of glycolysis – plays a key role in myeloma cell metabolism. Our findings reveal that myeloma cells predominantly rely on the uptake of pyruvate produced by neighbouring BMM-SCs via the plasma membrane proton-linked monocarboxylate transporters MCT-1 and MCT-2 encoded by the Slc16a1 and a2 genes, respectively. Furthermore, we show that pharmacological inhibition of the MCT-1/2, with AZD3965, triggers a cascade of compensatory metabolic responses, disrupting redox balance and significantly reducing the proliferation capacity of co-cultured myeloma cells.
{"title":"Pyruvate from bone marrow mesenchymal stem cells supports myeloma redox homeostasis and anabolism","authors":"Elías Vera-Sigüenza, Cristina Escribano-Gonzalez, Irene Serrano-Gonzalo, Kattri-Liis Eskla, Charlotte Speakman, Alejandro Huerta-Uribe, Lisa Vettore, Himani Rana, Adam Boufersaoui, Hans Vellama, Ramin Nashebi, Ielyaas Cloete, Jennie Roberts, Supratik Basu, Mark Drayson, Christopher Bunce, Guy Pratt, Fabian Spill, Oliver D.K. Maddocks, Daniel A. Tennant","doi":"10.1101/2024.08.08.607157","DOIUrl":"https://doi.org/10.1101/2024.08.08.607157","url":null,"abstract":"Multiple myeloma is an incurable cancer of plasma cells that depends on the bone marrow for its survival. Despite its prevalence, the molecular mechanisms underlying this malignancy remain poorly understood. In this study, we aim to bridge this knowledge gap by elucidating the metabolic interplay between myeloma cells and bone marrow mesenchymal stem cells (BMMSCs). BMMSCs are crucial in supporting myeloma cell metabolism, contributing to their proliferation, survival, and resistance to chemotherapy. Through a combination of mathematical modelling and experimental co-cultures, we demonstrate that pyruvate – the end product of glycolysis – plays a key role in myeloma cell metabolism. Our findings reveal that myeloma cells predominantly rely on the uptake of pyruvate produced by neighbouring BMM-SCs via the plasma membrane proton-linked monocarboxylate transporters MCT-1 and MCT-2 encoded by the Slc16a1 and a2 genes, respectively. Furthermore, we show that pharmacological inhibition of the MCT-1/2, with AZD3965, triggers a cascade of compensatory metabolic responses, disrupting redox balance and significantly reducing the proliferation capacity of co-cultured myeloma cells.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":"88 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141931018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Doxorubicin, a potent chemotherapeutic drug, is widely used against various cancers, notably breast cancer. While its efficacy is well-documented, precise dosage determination in experimental models remains challenging. Zebrafish xenografts of various cancers confirm doxorubicin's anti-cancerous effect; however, since doxorubicin treatment of zebrafish larva is done by adding doxorubicin to fish water, the precise chemotherapeutic dosage for zebrafish larva remains unknown. In this study, we provide a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for quantifying doxorubicin uptake in zebrafish larvae and thus provide a direct estimate of doses required for the therapeutic effect. Alongside quantification, we measured the therapeutic effect of doxorubicin in zebrafish larvae xenografted with triple negative breast cancer cell line, MDA-MB-231. LD50 value of doxorubicin was first determined by incubating 3-days post fertilization (dpf) larvae with different doses of doxorubicin for 72 h. Doxorubicin was quantified both from zebrafish larval homogenate and treatment solution. Analysis was performed by selected-reaction monitoring (SRM) scans in positive ionization mode. LD50 value for 72 h calculated to be 35.95 mg/L. As expected, doxorubicin-treated xenografts exhibited a significant reduction in tumor growth. The range of limit of detection (LOD) and limit of quantification (LOQ) for doxorubicin were 2 and 5 μg/L respectively. Intra- and inter-day accuracy was within the range of 82-114%. Overall, in this study we describe a reliable method for quantifying doxorubicin in zebrafish larvae. Our study facilitates precise dosage estimation, enhancing the relevance of zebrafish xenograft model in cancer research and potentially improving translational applications of chemotherapeutic treatments.
{"title":"Optimized LC-MS/MS method for Doxorubicin quantification: validating drug uptake and tumor reduction in zebrafish xenograft model of breast cancer","authors":"Ghazala Rahman, Atanu Pramanik, Susmita Das, Anindya Roy, Anamika Bhargava","doi":"10.1101/2024.08.09.607268","DOIUrl":"https://doi.org/10.1101/2024.08.09.607268","url":null,"abstract":"Doxorubicin, a potent chemotherapeutic drug, is widely used against various cancers, notably breast cancer. While its efficacy is well-documented, precise dosage determination in experimental models remains challenging. Zebrafish xenografts of various cancers confirm doxorubicin's anti-cancerous effect; however, since doxorubicin treatment of zebrafish larva is done by adding doxorubicin to fish water, the precise chemotherapeutic dosage for zebrafish larva remains unknown. In this study, we provide a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for quantifying doxorubicin uptake in zebrafish larvae and thus provide a direct estimate of doses required for the therapeutic effect. Alongside quantification, we measured the therapeutic effect of doxorubicin in zebrafish larvae xenografted with triple negative breast cancer cell line, MDA-MB-231. LD50 value of doxorubicin was first determined by incubating 3-days post fertilization (dpf) larvae with different doses of doxorubicin for 72 h. Doxorubicin was quantified both from zebrafish larval homogenate and treatment solution. Analysis was performed by selected-reaction monitoring (SRM) scans in positive ionization mode. LD50 value for 72 h calculated to be 35.95 mg/L. As expected, doxorubicin-treated xenografts exhibited a significant reduction in tumor growth. The range of limit of detection (LOD) and limit of quantification (LOQ) for doxorubicin were 2 and 5 μg/L respectively. Intra- and inter-day accuracy was within the range of 82-114%. Overall, in this study we describe a reliable method for quantifying doxorubicin in zebrafish larvae. Our study facilitates precise dosage estimation, enhancing the relevance of zebrafish xenograft model in cancer research and potentially improving translational applications of chemotherapeutic treatments.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141931016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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